title: Comparative immunochemical characteristics of botulinum neurotoxin type A and its associated proteins.
abstract: Clostridium botulinum strains secrete their neurotoxins (BoNT) along with a group of neurotoxin-associated proteins (NAPs) that enhance the oral toxicity and provide protection to the neurotoxin against acidity, temperature and proteases in the G.I. tract. A major component of NAPs is Hn-33, a 33 kDa protein, which is also protease resistant and strongly protects BoNT. The complex form of BoNT/A is used as a commercial therapeutic formulation against many neuromuscular disorders and for cosmetic purposes. Immune response against this formulation could hinder its long-term use; therefore, it is important to characterize the immunological properties of the associated proteins. This study aims to understand the immunological reactivity of BoNT/A complex, BoNT, NAPs, and Hn-33 through a series of competitive enzyme-linked immunosorbent assays (ELISA). The results indicated that BoNT/A complex competed 6 times more with complex antibodies compared to the neurotoxin confirming that the higher immunogenicity of BoNT/A complex was indeed a result of the associated proteins with the neurotoxin complex. While the nearly identical immuno-reactivity of BoNT/A complex and Hn-33 with Hn-33 antibodies indicated that the reactivity was due to the higher immunogenicity not the abundance of Hn-33 in the complex. Both the ELISA and immuno-blot results implied that Hn-33 is primarily responsible for eliciting the antibody response in BoNT/A complex.
authors.affiliation: Botulinum Research Center, Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Bryant
authors.forename: Shuowei
authors.initials: S
authors.lastname: Cai
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000502:physiology; D001667:Binding, Competitive; D001905:Botulinum Toxins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D003014:Clostridium botulinum / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D011817:Rabbits
chemical_list: D000906:Antibodies; C118973:hemagglutinin 33 protein, Clostridium botulinum; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A
keywords: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; ABTS; BoNT; BoNT/A; Botulinum; Clostridium; Complex; ELISA; Hn-33; IgG; Immunogenicity; NAPs; NBP; PBS; PBST; TBST; Tris buffered saline containing 0.05% Tween-20; botulinum neurotoxin; botulinum neurotoxin type A; enzyme-linked immunosorbent assay; immunoglobulin G; neurotoxin-associated proteins; neurotoxin-binding protein; phosphate buffered saline; phosphate buffered saline containing 0.05% Tween-20